Two IFN-&#945;s can bind to IFNAR2-EC as detected by ELISA. To examine the binding of two IFN species (CM3-6-histidine-tag and IFN-&#945;2c-6-histidine-tag) to the receptor subunit IFNAR2-EC, a competitive sandwich IFNAR2-EC ELISA was developed. We found that after saturation of IFNAR2-EC with CM3-6-histidine-tag, the receptor subunit was still able to bind IFN-&#945;2c-6-histidine-tag with low displacement of the CM3-6-Histidine tag. Using a surface plasmon resonance biosensor, we determined that the IFN-alpha hybrid CM3 stabilized IFN-&#945;2c/IFNAR2-EC complex. Analysis of IFN-&#945;2c and CM3 oligomeric state in solution showed that the sedimentation profile of IFN-&#945;2c was in sharp contrast to that of CM3. In order to gain more insight into the possible binding mechanisms, and the differences in receptor binding observed in SPR, the oligomeric state of both IFN molecules was studied by sedimentation velocity analytical ultracentrifugation. The rate of migration of the IFNs through a centrifugal field was monitored using analytical ultracentrifugation sedimentation velocity (AUC/SV). The results showed that the sedimentation profile of CM3 does not (or only very weakly) interact with IFN-&#945;2c in solution.[unreadable] Analysis of IFN-receptor complexes in solution confirmed the presence of two IFNs (IFN-&#945;2c-6-histidine-tag, CM3) and IFNAR2-EC. Native electrophoresis experiments showed the IFNs to be largely competitive, but also allowed detection of a population of triple complexes with both IFNs. IFNAR2-EC mutants suggest that CM3 and IFN-&#945;2c could use different amino acids within the binding domain of IFNAR2-EC. [unreadable] Differences between IFN-&#945;2c and CM3 in the antiproliferative assay are unlikely to be a consequence of interaction with IFNAR2. Neutralization of antiproliferative activities of IFNs on Daudi cells with soluble IFNAR2-EC show that IFNAR2-EC neutralized both IFNs to the same degree, suggesting that IFN-&#945;2c and CM3 are binding to IFNAR2-EC with similar affinity. This observation led to the conclusion that differences between IFN-&#945;2c and CM3 in the antiproliferative assay are not due to problems in interaction with IFNAR2.[unreadable] The N-terminal 6-histidine tag appears to affect the protein structure of IFN-&#945;2c. From the results of this study we could conclude that the N-terminal 6-histidine tag may affect protein structure and cause local misfolding and steric hindrance on the IFN-&#945;2c molecule. While we did not predict dramatic structural changes, the changes that occurred were sufficient to influence the recognition of the antibodies respective epitopes and to reduce specific antiviral and antiproliferative activities